J E Lafont

Hypoxia Promotes the Differentiated Human Articular Chondrocyte Phenotype through SOX9-dependent and -independent Pathways

The chondrocyte is solely responsible for synthesis and maintenance of the resilient articular cartilage matrix that gives this load-bearing tissue its mechanical integrity. When the differentiated cell phenotype is lost, the matrix becomes compromised and cartilage function begins to fail. We have recently shown that hypoxia promotes the differentiated phenotype through hypoxia-inducible factor 2α (HIF-2α)-mediated SOX9 induction of the main matrix genes. However, to date, only a few genes have been shown to be SOX9 targets, while little is known about SOX9-independent regulators. We therefore performed a detailed microarray study to address these issues. Analysis involved 35 arrays on chondrocytes obtained from seven healthy, non-elderly human cartilage samples. Genes were selected that were down-regulated with serial passage in culture (as this causes loss of the differentiated phenotype) and subsequently up-regulated in hypoxia. The importance of key findings was further probed using the technique of RNA interference on these human articular chondrocytes. Our results show that hypoxia has a broader beneficial effect on the chondrocyte phenotype than has been previously described. Of especial note, we report new hypoxia-inducible and SOX9-regulated genes, Gdf10 and Chm-I. In addition, Mig6 and InhbA were induced by hypoxia, predominantly via HIF-2α, but were not regulated by SOX9. Therefore, hypoxia, and more specifically HIF-2α, promotes both SOX9-dependent and -independent factors important for cartilage homeostasis. HIF-2α may therefore represent a new and promising therapeutic target for cartilage repair.

Type II Collagen Expression Is Regulated by Tissue-specific miR-675 in Human Articular Chondrocytes

miRNAs have been shown to be essential for normal cartilage development in the mouse. However, the role of specific miRNAs in cartilage function is unknown. Using rarely available healthy human chondrocytes (obtained from 8 to 50 year old patients), we detected a most highly abundant primary miRNA H19, whose expression was heavily dependent on cartilage master regulator SOX9. Across a range of murine tissues, expression of both H19- and H19-derived miR-675 mirrored that of cartilage-specific SOX9. miR-675 was shown to up-regulate the essential cartilage matrix component COL2A1, and overexpression of miR-675 rescued COL2A1 levels in H19- or SOX9-depleted cells. We thus provide evidence that SOX9 positively regulates COL2A1 in human articular chondrocytes via a previously unreported miR-675-dependent mechanism. This represents a novel pathway regulating cartilage matrix production and identifies miR-675 as a promising new target for cartilage repair.

Hypoxia Promotes the Production and Inhibits the Destruction of Human Articular Cartilage

OBJECTIVE To determine the effects of hypoxia on both anabolic and catabolic pathways of metabolism in human articular cartilage and to elucidate the roles played by hypoxia-inducible factors (HIFs) in these responses. METHODS Normal human articular cartilage from a range of donors was obtained at the time of above-the-knee amputations due to sarcomas not involving the joint space. Fresh cartilage tissue explants and isolated cells were subjected to hypoxia and treatment with interleukin-1α. Cell transfections were performed on isolated human chondrocytes. RESULTS Using chromatin immunoprecipitation, we found that hypoxia induced cartilage production in human tissue explants through direct binding of HIF-2α to a specific site in the master-regulator gene SOX9. Importantly, hypoxia also suppressed spontaneous and induced destruction of human cartilage in explant culture. We found that anticatabolic responses were predominantly mediated by HIF-1α. Manipulation of the hypoxia-sensing pathway through depletion of HIF-targeting prolyl hydroxylase-containing protein 2 (PHD-2) further enhanced cartilage responses as compared to hypoxia alone. Hypoxic regulation of tissue-specific metabolism similar to that in human cartilage was observed in pig, but not mouse, cartilage. CONCLUSION We found that resident chondrocytes in human cartilage are exquisitely adapted to hypoxia and use it to regulate tissue-specific metabolism. Our data revealed that while fundamental regulators, such as SOX9, are key molecules both in mice and humans, the way in which they are controlled can differ. This is all the more important since it is upstream regulators such as this that need to be directly targeted for therapeutic benefit. HIF-specific hydroxylase PHD-2 may represent a relevant target for cartilage repair.

Lack of oxygen in articular cartilage: consequences for chondrocyte biology

Controlling the chondrocytes phenotype remains a major issue for cartilage repair strategies. These cells are crucial for the biomechanical properties and cartilage integrity because they are responsible of the secretion of a specific matrix. But chondrocyte dedifferentiation is frequently observed in cartilage pathology as well as in tissue culture, making their study more difficult. Given that normal articular cartilage is hypoxic, chondrocytes have a specific and adapted response to low oxygen environment. While huge progress has been performed on deciphering intracellular hypoxia signalling the last few years, nothing was known about the particular case of the chondrocyte biology in response to hypoxia. Recent findings in this growing field showed crucial influence of the hypoxia signalling on chondrocytes physiology and raised new potential targets to repair cartilage and maintain tissue integrity. This review will thus focus on describing hypoxia‐mediated chondrocyte function in the native articular cartilage.

Hypoxia. HIF-mediated articular chondrocyte function: prospects for cartilage repair

In a chronically hypoxic tissue such as cartilage, adaptations to hypoxia do not merely include cell survival responses, but also promotion of its specific function. This review will focus on describing such hypoxia-mediated chondrocyte function, in particular in the permanent articular cartilage. The molecular details of how chondrocytes sense and respond to hypoxia and how this promotes matrix synthesis have recently been examined, and specific manipulation of hypoxia-induced pathways is now considered to have potential therapeutic application to maintenance and repair of articular cartilage.